US7176035B2 - Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction - Google Patents

Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction Download PDF

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US7176035B2
US7176035B2 US10/010,310 US1031001A US7176035B2 US 7176035 B2 US7176035 B2 US 7176035B2 US 1031001 A US1031001 A US 1031001A US 7176035 B2 US7176035 B2 US 7176035B2
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Elias Georges
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GEORGES PHD ELIAS
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to proteomics. More specifically, the invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction.
  • Protein-protein interactions are critical events in biological processes. Protein-protein interactions govern biological processes that handle cellular information flow and control cellular decisions (e.g., signal transduction, cell cycle regulation and assembly of cellular structures).
  • the entire network of interactions between cellular proteins is a biological chart of functional events that regulate the internal working of living organisms and their responses to external signals.
  • a necessary step for the completion of this biological interaction chart is the knowledge of all the gene sequences in a given living organism.
  • the entire DNA sequence of the Homo sapiens genome will be completed at the latest by the year 2003 (112). Unfortunately, the sequence of a gene does not reveal its biological function nor its position in the biological chart. Given the expected number of proteins in the human genome (80,000 to 120,000), the mapping of the biological chart of protein-protein interactions will be an enormous but a rewarding task.
  • transcription of reporter genes depends on an interaction between a DNA-bound “bait” protein and an activation-domain containing “prey” protein.
  • the two hybrid systems unfortunately may suffer from a number of disadvantages. For example, the interaction of proteins is monitored in the nuclear milieu rather than the cytoplasm where most proteins are found and it does not allow the simultaneous identification of the precise amino acid sequences between two interacting proteins and cannot be easily applied to different cell types or tissues whereby different interacting proteins may be expressed.
  • the present invention seeks to meet these and other needs.
  • the present invention seeks to overcome the drawbacks of the prior art. More specifically, the invention concerns an approach to identify protein-protein interaction domains which differ from the prior art. Moreover, one approach of the present invention is based on an understanding of the principle that governs protein-protein interactions. Such understanding therefore, allows the use of several methods. Such a method is exemplified in detail below to identify: i) at least one of the exact amino acid sequences between interacting proteins; ii) a number of, possibly all, interacting proteins in different cells or tissues; and iii) the specific domains (or sequences) between two interacting proteins as targets for isolation of lead drugs. Preferably, the method and assay of the present invention enable a determination of i), ii) and iii). Moreover, unlike the approaches of the prior art, the method described herein allows for the identification of interacting proteins and the precise amino acid sequences of interactions in several days as opposed to several months.
  • the present invention further relates to a novel approach in drug discovery.
  • a major obstacle in drug development for the treatment of diseases has been the identification of target proteins and their functional sites.
  • R&D research and development
  • drugs that bind to and inhibit the functions of these proteins takes several years and is generally non-specific and random.
  • drugs identified by current approaches often target the active sites in proteins. Such drugs thus often lead to major side-effects. Therefore, it is not surprising that many R&D projects never lead to the development of specific drugs even after three to five years of intensive research efforts.
  • the methods and assays to identify protein-protein interactions of the present invention may address three important steps in the development of drugs:
  • P-glycoprotein has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs.
  • Analysis of P-gp amino acid sequence has lead to a proposed model of a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of about 90 amino acids, the linker domain.
  • similarly charged domains are found in other members of the P-gp superfamily, the function(s) of this domain are not known.
  • this domain binds to other cellular proteins.
  • a method of identifying a high affinity interacting domain in a chosen protein, domain thereof, or part thereof, and the amino acid sequence thereof comprising: a) providing a set of overlapping peptides spanning a complete sequence of the chosen protein, domain thereof, or part thereof, covalently bound to a support; b) providing a mixture of proteins and/or a mixture of peptides; c) incubating the set of overlapping peptides of a), with the mixture of b), under conditions enabling the binding between a high affinity interacting domain in a peptide of the set and one or more protein or peptide of b) to occur; d) washing off any protein-protein interaction which is not a high affinity interaction of c); and e) identifying which peptide of a) interacts with high affinity to a protein or peptide of b), thereby identifying the peptide of e) and the sequence thereof as a high affinity interacting domain.
  • a method of identifying an agent which modulates an interaction between high affinity interacting domains between a set of overlapping peptides spanning a complete sequence of a chosen protein, domain thereof or part thereof, covalently bound to a support and a mixture of proteins and/or a mixture of peptides comprising: a) incubating the set of overlapping peptides, with the mixture in the presence of at least one agent, under conditions enabling the binding between a high affinity interacting domain in a peptide of the set and one or more protein or peptide of the mixture to occur; b) washing off any protein-protein interaction which is not a high affinity interaction of b); and c) identifying which peptide of a) interacts with high affinity to a protein or peptide of the mixture in a presence of the agent as compared to in an absence thereof; thereby identifying the agent as a modulator of the high affinity interaction when the interaction in the presence of the agent is measurably
  • agents identified as modulators of the high affinity protein interactions of the present invention there is provided agents identified as modulators of the high affinity protein interactions of the present invention.
  • overlapping peptides spanning a peptide sequence refers to peptides of a chosen size, based on the sequence of the protein (or part thereof).
  • these peptides are synthetic peptides.
  • the size of the overlapping peptides has a significant impact on the workings of the present invention.
  • peptides of four contiguous amino acids appear to significantly increase the low affinity binding of proteins thereto.
  • larger peptides such as 20 amino acids or higher, would be expected to increase the proportion of repulsive amino acids to high affinity amino acids, thereby masking or totally inhibiting the binding of specific proteins to the peptides.
  • the preferred size for the overlapping peptides of the present invention is between 5 and 15 amino acids, more preferably between 5 and 12, and especially preferably between 5 and 10 amino acids.
  • support in the context of a support to which the overlapping peptides of the present invention are covalently bound, can be chosen from a multitude of supports found in the art. Such supports include CHIPS, plates (e.g. 96-well plates), glass beads and the like.
  • CHIP technology is well-known in the art (10, 19, 24, 26, 85, 97).
  • Protein sequences are presented herein using the one letter or three letter amino acid symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
  • rDNA recombinant DNA
  • nucleic acid molecule refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic DNA, cDNA) and RNA molecules (e.g., mRNA). The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]).
  • recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering.
  • DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • the nucleic acid e.g. DNA or RNA
  • the nucleic acid may be obtained according to well known methods.
  • physiologically relevant is meant to describe interactions which can take effect to modulate an activity or level of one or more proteins in their natural setting.
  • DNA molecule or sequence (as well as sometimes the term “oligonucleotide”) refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a “regulatory element” according to the present invention, as the term is defined herein.
  • oligonucleotide” or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5′ to 3′ direction.
  • Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (4, 96) and are commonly known in the art. In the case of hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65° C.
  • RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (96).
  • Probes for nucleic acids can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like. Modified sugar-phosphate backbones are generally taught (73, 75). Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the detection of the interaction between proteins and/or peptides be dependent on a label.
  • labels provide sensitivity and often enable automation.
  • automation is performed using CHIP technology.
  • the overlapping peptides spanning a chosen sequence of a protein are bound to a CHIP which can then be used to automate a test for interaction with proteins or peptides.
  • the present invention is not strictly dependent on a design and synthesis of the overlapping set of peptides spanning a chosen protein sequence. Indeed, banks of peptides are available, from which this set of overlapping peptides could be constructed.
  • Protein labelling is well known in the art.
  • labels include 3 H, 14 C, 32 P, and 35 S.
  • detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the protein.
  • the identification of the interaction is not specifically dependent on labelling of the proteins, since for example, this interaction could be assessed using proteomic approaches (such as 2-D gels and mass spectrometry) or using a library of antibodies.
  • radioactive amino acids can be incorporated into peptides or proteins of the invention by several well-known methods.
  • a non-limiting example thereof includes in vitro or in vivo labelling of proteins using 35 SMet.
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
  • expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA then being translated (translation) into one polypeptide (or protein) or more.
  • expression vector defines a vector or vehicle as described above, but designed to enable the expression of an inserted sequence following transformation into a host.
  • the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
  • control element sequences such as promoter sequences.
  • the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
  • Operably linked sequences may also include two segments that are transcribed into the same RNA transcript.
  • two sequences such as a promoter and a “reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • a promoter and a “reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • Prokaryotic expression is useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest.
  • This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e.g., SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography, etc.).
  • the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies. The purified protein can be used for therapeutic applications.
  • the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which in turn is operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.
  • the promoter is bound at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CCAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the ⁇ 10 and ⁇ 35 consensus sequences.
  • the designation “functional derivative” denotes, in the context of a functional derivative of a sequence, whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
  • This functional derivative or equivalent may be a natural derivative or may be prepared synthetically.
  • Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained.
  • the substituting amino acid When relating to a protein sequence, the substituting amino acid has chemico-physical properties, which are similar to that of the substituted amino acid.
  • the similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity and the like.
  • the term “functional derivatives” is intended to include fragments, segments, variants, analogs or chemical derivatives of the subject matter of the present invention.
  • a “conservative mutation or substitution” of an amino acid refers to mutation or substitution which maintains: 1) the structure of the backbone of the polypeptide (e.g. a beta sheet or alpha-helical structure); 2) the charge or hydrophobicity of the amino acid; or 3) the bulkiness of the side chain. More specifically, the well-known terminologies “hydrophilic residues” relate to serine or threonine. “Hydrophobic residues” refer to leucine, isoleucine, phenylalanine, valine or alanine. “Positively charged residues” relate to lysine, arginine or histidine. “Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to phenylalanine, tryptophan or tyrosine.
  • Peptides, protein fragments, and the like in accordance with the present invention can be modified in accordance with well-known methods dependently or independently of the sequence thereof.
  • peptides can be derived from the wild-type sequence exemplified herein in the figures using conservative amino acid substitutions at 1, 2, 3 or more positions.
  • conservative amino acid substitutions is well known in the art, which relates to substitution of a particular amino acid by one having a similar characteristic (e.g. aspartic acid for glutamic acid, or isoleucine for leucine).
  • non-conservative amino acid substitutions can also be carried out, as well as other types of modifications such as deletions or insertions, provided that these modifications modify the peptide, in a suitable way (e.g. without affecting the biological activity of the peptide if this is what is intended by the modification).
  • a list of exemplary conservative amino acid substitutions is given hereinbelow.
  • modifications of the peptides can also be effected without affecting the primary sequence thereof using an enzymatic or chemical treatment well known in the art.
  • variant refers herein to a protein or nucleic acid molecule, which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
  • the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology, using methods well known in the art.
  • a variant according to the present invention can be identified using a method of the present invention. It can also be designed to formally test for the conservation of particular amino acids (e.g. by synthesizing a variant or mutant peptide). These variants can also be tested as part of the full-length sequence of the protein in order to validate the interaction.
  • chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (e.g. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington's Pharmaceutical Sciences (88). Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art.
  • allele defines an alternative form of a gene, which occupies a given locus on a chromosome.
  • a “mutation” is a detectable change in the genetic material, which can be transmitted to a daughter cell.
  • a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
  • Spontaneous mutations and experimentally induced mutations exist.
  • the result of a mutation of a nucleic acid molecule is a mutant nucleic acid molecule.
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
  • purified refers to a molecule having been separated from a cellular component.
  • a “purified protein” has been purified to a level not found in nature.
  • a “substantially pure” molecule is a molecule that is lacking in most other cellular components.
  • molecule As used herein, the terms “molecule”, “compound” or “ligand” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds.
  • the term “molecule” therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
  • Non-limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents.
  • the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling, combinatorial library screening and the like.
  • the terms “rationally selected” or “rationally designed” are meant to define compounds, which have been chosen based on the configuration of the interaction domains of the present invention.
  • molecules having non-naturally occurring modifications are also within the scope of the term “molecule”.
  • peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs
  • the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain.
  • the molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by a high affinity protein interaction identified in accordance with the present invention.
  • the molecules identified in accordance with the teachings of the present invention find utility in the development of more efficient agents, which can modulate such interactions.
  • Libraries of compounds are well known in the art. Libraries of peptides are also available. Such libraries can be used to build an overlapping set of peptide sequences spanning a chosen domain, protein or part thereof.
  • indicator cells refers to cells that express, in one particular embodiment, two interacting peptide domains of the present invention, and wherein an interaction between these proteins or interacting domains thereof is coupled to an identifiable or selectable phenotype or characteristic such that it provides an assessment or validation of the interaction between same.
  • Such indicator cells can also be used in the screening assays of the present invention.
  • the indicator cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of these interacting domains.
  • the cells can be yeast cells or higher eukaryotic cells such as mammalian cells (WO 96/41169).
  • the indicator cell is a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (4) and can be used to test a compound or a library thereof.
  • a reporter gene encoding a selectable marker or an assayable protein can be operably linked to a control element such that expression of the selectable marker or assayable protein is dependent on the interaction of the Protein A and Protein B interacting domains.
  • the reporter gene is luciferase or ⁇ -Gal.
  • At least one of the two interacting proteins or domains of the present invention may be provided as a fusion protein.
  • the design of constructs therefor and the expression and production of fusion proteins are well known in the art (4, 96).
  • both interaction domains are part of fusion proteins.
  • a non-limiting example of such fusion proteins includes a LexA-Protein A fusion (DNA-binding domain-Protein A; bait) and a B42-Protein B fusion (transactivator domain-Protein B; prey).
  • the LexA-Protein A and B42-Protein B fusion proteins are expressed in a yeast cell also harboring a reporter gene operably linked to a LexA operator and/or LexA responsive element.
  • fusion proteins can be used in such two hybrid systems.
  • the fusion proteins need not contain the full-length interacting proteins. Indeed, fragments of these polypeptides, provided that they comprise the interacting domains, can be used in accordance with the present invention, as evidenced with the peptide spanning method of the present invention.
  • Non-limiting examples of such fusion proteins include hemagglutinin fusions, gluthione-S-transferase (GST) fusions and maltose binding protein (MBP) fusions.
  • GST gluthione-S-transferase
  • MBP maltose binding protein
  • protease cleavage sites between two heterologously fused polypeptides are well known in the art.
  • the interaction domains of the present invention it might also be beneficial to fuse the interaction domains of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell.
  • Signal peptides from diverse organisms are well known in the art.
  • Bacterial OmpA and yeast Suc2 are two non limiting examples of proteins containing signal sequences.
  • linker commonly known
  • sequences and polypeptides useful to practice the invention include, without being limited thereto, mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) domain. It will be clear to the person of ordinary skill that whether an interaction domain, variant, derivative, or fragment thereof, of the present invention retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
  • the interaction domains of the present invention can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds.
  • some derivative or analogs having lost their biological function of interacting with their respective interaction partner may still find utility, for example for raising antibodies.
  • Such analogs or derivatives could be used for example to raise antibodies to the interaction domains of the present invention.
  • These antibodies could be used for detection or purification purposes.
  • these antibodies could also act as competitive or non-competitive inhibitors and be found to be modulators of an interaction identified in accordance with the present invention.
  • a host cell or indicator cell has been “transfected” by exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has been introduced inside the cell.
  • the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transfecting DNA may be maintained on a episomal element such as a plasmid.
  • a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • the present invention also provides polyclonal, monoclonal antibodies, or humanized versions thereof, chimeric antibodies and the like which inhibit or neutralize their respective interaction domains and/or are specific thereto.
  • the term “therapeutic agent” should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents.
  • the DNA segments or proteins according to the present invention can be introduced into individuals in a number of ways. For example, erythropoietic cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways, including intravenous injection. Alternatively, the DNA construct can be administered directly to the afflicted individual, for example, by injection in the bone marrow.
  • the therapeutic agent can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes.
  • the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (e.g. DNA construct, protein, molecule), the response and condition of the patient as well as the severity of the disease.
  • the chosen therapeutic regimen e.g. DNA construct, protein, molecule
  • compositions within the scope of the present invention should contain the active agent (e.g. protein, nucleic acid, or molecule) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects.
  • the nucleic acids in accordance with the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated.
  • Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (88).
  • the amount administered should be chosen so as to avoid adverse side effects.
  • the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
  • FIG. 1 shows the principle of protein-protein interaction.
  • the plus sign (+) indicate the regions of high affinity binding.
  • the minus signs ( ⁇ ⁇ ) indicate the regions of high-repulsive forces.
  • interactions between two proteins are made up of discontinuous regions of high affinity binding and high-repulsive forces that are almost in equilibrium with high affinity binding being more favoured while proteins are together.
  • FIG. 2 is a schematic representation of a method of identification of high affinity binding sequences according to one embodiment of the present invention.
  • 2 A the different shapes represent different proteins in a total cell lysate. The signs are like for FIG. 1.
  • 2B small overlapping peptides that cover the entire sequence (or a segment) of protein. A will be synthesized directly on derivatized wells of 96-well polypropylene plates. Following peptide synthesis, metabolically radiolabeled total cell lysate is added to each well containing the various peptides and incubated in an incubator buffer.
  • the dark filled circles represent the radiolabeled proteins from total cell lysate isolated from metabolically radiolabeled cells added to all the wells of the 96-well plates to identify high affinity binding sequences on Protein A.
  • the high affinity binding sequences (overlapping peptides from Protein A) are in those wells that bind radiolabeled proteins (in dark).
  • Four high affinity binding sequences between Protein A and another protein(s) are identified in rows 1, 3, 6 and 8.
  • the wells that contain the high affinity binding sequences are identified by radiolabeled counting and SDS-PAGE.
  • FIG. 3 is a schematic representation of a method of identification of high affinity binding sequences according to another embodiment of the present invention.
  • 3 A shows a schematic representation of the interaction between Protein A and Protein B.
  • 3 B small overlapping peptides that cover the entire sequence (or a segment) of Protein A will be synthesized directly on derivatized wells of 96-well polypropylene plates. Following peptide synthesis, a radiolabelled Protein B (synthesized from in vitro transcription-translation reaction mix) are added to each well containing the various peptides and incubated in an incubation buffer.
  • 3 C the dark filled circles represent the radiolabeled Protein B that has been added to all the wells of the 96-well plates to identify high affinity binding sequences on Protein A.
  • 3 D after a washing procedure, the high affinity binding sequences are in those wells in which Protein B (radiolabeled protein in dark) is still bound to the peptides from Protein A.
  • FIG. 4 is a schematic representation of a method of selection of drugs that specifically inhibit the binding of protein A to B according to one embodiment of the present invention.
  • 4 A shows a schematic representation of the interaction between Protein A and Protein B.
  • 4 B peptides that encode high affinity binding sequences are used as LEAD sequences for the selection of specific drugs that inhibit the association between Protein A and Protein B and ultimately the function of the complex.
  • peptides encoding one of the high affinity binding sequences are synthesized in every well of the 96-well plate.
  • Grey circles represent one of four high affinity binding sequences identified in FIGS. 2 and 3 .
  • radiolabeled Protein B are added to each of the wells.
  • combinatorial libraries can be screened to identify drugs that bind specifically to the high affinity binding sequences of Protein A.
  • radiolabeled Protein B from transcription-translation reaction mix are represented. Plates are washed and drugs that specifically bind to high affinity sequences of Protein A are found in those wells that do not contain radiolabeled Protein B.
  • wells containing drugs/compounds that bind specifically to one of the high affinity binding sequence in Protein A and therefore prevent the binding of Protein B are identified by the absence of a dark circle (i.e., wells 28, 70 and 75).
  • Selected drugs/compounds represent invaluable LEAD compounds that can be used in biological assays to confirm their mechanism of action. Validated drugs can proceed toward in vivo studies.
  • FIG. 5 shows a P-glycoprotein predicted secondary structure and amino acid of the linker domain.
  • the twelve filled squares represent the twelve putative transmembrane domains.
  • the two ATP binding domains are represented by two circles in the N- and C-terminal halves of P-gp.
  • the inset represents the linker domain.
  • the amino acid sequence of the linker domains of Human P-gp 1 (HP-gp1) and HP-gp3 is indicated as a single-letter amino acid code.
  • each amino acid sequence of HP-gp1 (SEQ ID NO: 15) and HP-gp3 (residues 1–89 of SEQ ID NO: 14) shows the length of the linker domains (1–90 and 1–89 for HP-gp1 and HP-gp3, respectively).
  • the numbered lines underneath the amino acid sequence show the sequences of the overlapping hexapeptides, which differ by one amino acid. For HP-gp3, the last hexapeptide is number 89.
  • FIGS. 6A–C shows the protein binding to overlapping hexapeptides encoding HP-gp1 linker domain. Overlapping hexapeptides that encode the linker domain of HP-gp1 were synthesized on polypropylene rods and used to identify proteins that bind to these peptides. A total of 90 plus two control hexapeptides for HP-gp1 were incubated with total cell lysate from [ 35 S] methionine metabolically labeled cells (see methods). All bound proteins were eluted from the peptide-fixed rods and resolved on 10% SDS-PAGE. Lanes 1 to 92 show the [ 35 S] methionine bound proteins from HP-gp1. The migration of the molecular weight markers is shown to the left of gels.
  • FIG. 7 shows the effects of different detergents or high salt on the binding of proteins to HP-gp1 hexapeptides.
  • Metabolically radiolabeled proteins bound to hexapeptides (hexapeptides 50 to 53) from HP-gp1 linker domain were eluted in the presence of increasing concentrations of anionic detergent (0.12%–0.5% SDS), zwitterionic detergent (20 mM–80 mM CHAPS) or salt (0.3 M–1.2 M KCl).
  • the y-axis represents the amount of radioactivity eluted from a pool of three hexapeptides (50 to 53).
  • FIG. 8 shows the effects of CHAPS on the binding of proteins to the overlapping hexapeptides encoding HP-gp1 linker domain.
  • Overlapping hexapeptides of the linker domain of HP-gp1 were incubated with total cell lysate from [ 35 S] methionine metabolically labeled cells extracted with 10 mM CHAPS.
  • Bound proteins were eluted from the peptide-fixed rods and resolved by 10% SDS-PAGE.
  • Lanes 1 to 92 show the [ 35 S] methionine bound proteins to HP-gp1 linker domain. The migration of the molecular weight markers is shown to the left of gels.
  • FIG. 9 shows the protein binding to overlapping hexapeptides encoding HP-gp3 linker domain. Overlapping hexapeptides that encode the linker domain of HP-gp3 were synthesized on polypropylene rods and used to identify proteins that bind to these peptides. A total of 88 plus two control hexapeptides for HP-gp3 were incubated with total cell lysate from [ 35 S] methionine metabolically labeled cells. All bound proteins were eluted from the peptide-fixed rods and resolved on 10% SDS-PAGE. Lanes 1 to 90 show the [ 35 S] methionine bound proteins from HP-gp3. The migration of the molecular weight markers is shown to the left of gels.
  • FIG. 10 shows the sequence alignment of three binding regions of HP-gp1 and HP-gp3 linker domains. Alignment of HP-gp1 (SEQ ID NO: 15) and HP-gp3 (SEQ ID NO: 14) linker domains is shown using a single-letter code for amino acids. The regions of high binding affinities for HP-gp3 and HP-gp1 are shown in bold. Identical amino acids are shown by single letter code between the two aligned sequences. conserveed amino acids are indicated by plus (+) sign. The numbers on each side of the amino acid sequence of the linker domains refer to the amino acid sequence of human P-gp1 and 3 as in (90, 111).
  • FIG. 11 shows the two high affinity binding hexapeptides.
  • Two high affinity binding sequences 658 RSSLIR 663 (SEQ ID NO: 7) and 669 SVRGSQ 674 (SEQ ID NO: 8) from HP-gp1 linker domain were resynthesized and incubated with total cell lysate from [ 35 S] methionine metabolically labeled cells following 24 hour or 48 hour incubation times. Bound proteins were eluted from peptide-fixed rods and resolved by 10% SDS-PAGE. The migration of the molecular weight markers is shown to the left of the figure.
  • FIG. 12 shows the effects of different carrier proteins as blocking agent of unspecific binding.
  • Total cell lysates from [ 35 S] methionine metabolically labeled CEM cells were used as is or made 1% gelatin, 0.3% BSA or 3% BSA.
  • the cell lysates were incubated with a high affinity binding hexapeptide 658 RSSLIR 663 (SEQ ID NO: 7) from HP-gp1 linker domain.
  • the bound proteins were eluted with SDS sample buffer and resolved on 10% SDS-PAGE. The migration of the molecular weight markers is shown to the left of the figure.
  • FIG. 13 shows the purification of a 57 kDa protein.
  • Total cell lysate was incubated with fifty HP-gp1 hexapeptides 658 RSSLIR 663 (SEQ ID NO: 7) and 669 SVRGSQ 674 (SEQ ID NO: 8).
  • Samples containing the 57 kDa protein (P57) from one hundred hexapeptide incubation mix were pooled and resolved by 10% SDS-PAGE. The resolved proteins were transferred to PVDF membrane and stained with Ponceau S. The migration of the molecular weight markers is shown to the right of the figure.
  • FIG. 14 shows a Western blot analysis with anti-tubulin monoclonal antibodies.
  • Total cell lysate from CEM cells and proteins eluted from the high affinity binding hexapeptides of HP-gp1 linker domain (P57) were resolved on SDS-PAGE and transferred to nitrocellulose membrane.
  • One half of the membrane was probed with anti- ⁇ and anti- ⁇ tubulin monoclonal antibodies. The migration of the molecular weight markers is shown to the left of the figure.
  • FIG. 15 shows the helical wheel presentations of the high affinity binding region of HP-gp1 and HP-gp3 linker domains.
  • the single-letter amino acid code for the high affinity binding region of HP-gp1 (SEQ ID NO: 12) and HP-gp3 (SEQ ID NO: 13) linker domains are shown.
  • the positively charged amino acids on one side of the helix have been circled.
  • proteins The function or functions of proteins is mediated through an interaction thereof with other cellular or extracellular proteins.
  • interactions between two proteins involve large segments of polypeptides that have complementary amino acid sequences.
  • these complementary sequences mediate the interactions between proteins.
  • a novel concept to explain the principle of protein-protein interactions is proposed. Briefly, interactions between any two or more proteins are mediated by strings of discontinuous sequences with high affinity binding and high-repulsive forces (see FIG. 1 ). The sum of these forces over the entire exposed sequence of proteins determines the nature and extent of the interactions between proteins.
  • the sizes of these interacting domains can vary from 5 to 25 amino acids in length.
  • the attractive forces between two small high affinity binding sequences are generally larger than the sum of all the high affinity binding and repulsive-forces between two proteins. Therefore, using the present approach, it is possible to isolate interacting proteins from a mixture of proteins using a short peptide (almost six amino acids) that encodes only the high affinity binding sequence. Indeed, with this in mind, it is now easy to see why many methods attempting to isolate interacting proteins have failed. The use of large fragments or proteins to isolate interacting proteins is less efficient since the sum of attractive/repulsive forces are much weaker than any string of attractive forces.
  • Non-limiting examples of such small consensus sequences include the leucine zipper (63), and SH2 and SH3 binding sequences (63, 80).
  • protein-protein interactions can have many measurable effects, such as: i) changes in the kinetic properties of one or both proteins (83, 84); ii) formation of new binding or functional sites (65, 104); and iii) the inactivation of function(s) (106, 114).
  • a given protein could expose different functional domains or sequences in the presence (as opposed to the absence) of any interacting proteins.
  • protein A in the presence of protein B, protein A can expose other sequences not previously exposed for interactions with other proteins (65, 83, 84, 104, 106, 114).
  • the latter concept is very important as it argues against the effectiveness of some structural studies (i.e., X-ray and NMR) in predicting functional or surface exposed domains from the resolved crystal structure of proteins.
  • some structural studies i.e., X-ray and NMR
  • the present invention seeks to overcome the drawbacks of the results obtained from such structural studies.
  • dimerization a subset of protein-protein interactions is dimerization.
  • homo- or heterodimers include; growth factor receptors (52); membrane transport proteins (9, 36, 76); tumor suppressor proteins (72); and proteins that mediate apoptosis (87).
  • dynamic dimerization is a common theme in the regulation of signal transduction.
  • Some of the functional consequences of dimerization include, increased proximity for activation of single transmembrane cell surface receptors (e.g., EGF receptor (52)) and differential regulation by heterodimerization [e.g., BCL2 family of proteins (87)].
  • the protein concentration in living cells is very high and is in the range of 10–30 mg/ml. At this high protein concentration, most if not all proteins should interact precisely and specifically with other cellular proteins. Some of the interacting proteins act as inhibitors of function, while others may be activators (e.g., The BCL2-BAX family of proteins, (87)). Moreover, the cycling of a given protein between activator and inhibitor association will require the association-dissociation process to occur rapidly. For example, when protein X is associated with an inhibitor protein I, the domains (small sequences) that are required for the association of protein X with an activator protein A may not be easily accessible in the X-I complex.
  • the method of the present invention is capable of identifying all functional domains or high affinity interacting domains of protein X and its associated proteins. Once the associated proteins are identified, their biological functions as it relates to the target protein X can be tested.
  • the high affinity binding sequences (between protein X and Protein I or A) can be easily identified and can be used as a target site in a high throughput drug screening assays (see below) or other assays.
  • This invention includes the concept (described in FIGS. 1A–1D ) that protein-protein interactions are made-up of discontinuous high affinity binding and high-repulsive forces scattered throughout the 3-D sequence of proteins and that these sequences can be isolated using one of many possible approaches indicated herein (e.g., the overlapping peptide approach). Although, in this application, the overlapping peptide approach is exemplified, other approaches can be envisioned that give similar results. It should be stressed that the approach described herein is immune to conformational changes resulting from interacting proteins that could effect other commonly used methods to identify protein-protein interactions (e.g., two-hybrid system, affinity blotting, and crosslinking).
  • Protein A is fused with another protein sequence (the DNA-bound “bait” protein) and the other interacting protein is fused to the activation-domain containing “prey” protein.
  • the fusion of interacting proteins to protein A could expose regions other than those found in the native conformation which will affect their interactions.
  • the two-hybrid system has several disadvantages, some of which are listed below,
  • Proteins can be toxic when expressed in different cells or organisms.
  • the post-translational modification of one protein can exclude its interaction with other proteins.
  • the two-hybrid system does not allow the simultaneous identification of the precise amino acid sequences between two interacting proteins.
  • the two-hybrid system cannot be easily applied to different cell types or tissues whereby different interacting proteins may be expressed (this can be a critical drawback of this system).
  • the present approach and methodology used to identify discontinuous strings of sequences between two or more interactive proteins is a scanning overlapping peptide approach.
  • a large number of short overlapping peptides which cover the entire amino acid sequence of the given protein, “the bait,” are synthesized in parallel on an inert solid support (see FIG. 2 ).
  • the rationale for synthesizing a large number of overlapping peptides as opposed to a discontinuous peptide library is based on the fact that one does not know a priori what exact sequence of a given protein will contain the high affinity binding sites and the repulsive sequences. Therefore, a discontinuous peptide approach will often lead to the presence of both high affinity binding sequences and repulsive sequences in the same peptide.
  • peptides will not bind to potential interacting proteins with high affinity.
  • the use of overlapping peptides also provides internal controls for unspecific binding.
  • the high affinity binding sequences will give a peak of signal when peptides within the high affinity domain will have the high affinity amino acid sequences but will lack amino acids which provide the repulsive forces (see FIG. 6 in Example I).
  • the present invention is not dependent on a spanning of the full peptide sequence. Indeed, sub-region(s) of a protein can be used.
  • overlapping peptides can be derived from a chosen domain of a protein. Also, it would be envisageable to probe an overlapping peptide side set of a first protein with an overlapping peptide set of a second protein.
  • P-glycoprotein is a membrane protein (46) that confers resistance to anticancer drugs and therefore is responsible for the failure of chemotherapy.
  • P-glycoprotein has been shown to function by preventing the accumulation of chemotherapeutic drugs in tumor cells; the exact mechanism of how this protein functions and what are the associated proteins that modulate its function are not known.
  • the peptides and total cell lysate were incubated in the presence of a carrier substrate (1–3% bovine serum albumin, or 1–3% gelatin, 1–3% skim milk, etc.) for 18 hours at 4° C. Following this incubation period, the covalently fixed peptides were washed extensively with isotonic buffer. Any proteins from the radiolabeled total cell lysate which maintained their association with the overlapping hexapeptides following the washing step are eluted in SDS-contain sample buffer and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) (62). The presence of radiolabeled proteins on SDS polyacrylamide gels following gel drying and signal enhancement, provides the following information:
  • those specific overlapping peptides represent high affinity binding sequences in the P-glycoprotein linker domain (or other chosen domains or non-chosen domains);
  • the associated proteins which bound to the high affinity binding sequences can be isolated in large quantities for the purpose of determining their identity by N-terminal amino acid sequencing by Edman degradation (27) or the like. Briefly, the sequences of the overlapping peptides that bound a given protein are resynthesized on a solid support and kept fixed thereto. Total cell lysate from [ 35 S]methionine metabolically-radiolabeled cells is added to the solid support containing the fixed high affinity sequence peptides and incubated as described above. Following washing steps to remove unbound material, the associated protein is isolated in large amounts following an elution step with SDS-containing buffers (see below). The purified associated protein is now ready for amino acid sequencing.
  • the same concept as described above can be applied if one is only interest in identifying the high affinity binding sequences between two proteins.
  • a non-limiting example of such two proteins are the regions of interactions between p53 and MDM (28, 103).
  • the purpose of this exercise is to identify the high affinity binding sequences between proteins A (p53) and protein B (MDM) in order to use these sequences as target sites for the identification of compounds that modulate this interaction and more particularly for the development of drugs.
  • a given drug when a given drug is bound to one of these high affinity binding sites on protein A, it will prevent the formation of the active complex (protein A+B) and therefore inhibit the functions of the complex.
  • To isolate the string of high affinity binding sequences between Protein A and B see FIG.
  • the peptides encoding protein A are incubated with radiolabeled protein B to allow for binding to occur. Following an incubation period (5 to 24 hours), unbound radiolabeled protein B will be removed by extensive washing in isotonic buffer. Any overlapping peptides which bound to radiolabeled protein B will be eluted in the presence of denaturing agents. The eluant from each of the 96-well plates are analyzed for the presence of radiolabeled protein B by running the samples by SDS-PAGE (62). High affinity binding peptides will be identified as those that retain the radiolabeled Protein B.
  • metabolically radiolabeled proteins as “the prey” to interact with the overlapping peptides of “the bait” increases the sensitivity of this technique and allows the identification of interacting proteins with binding affinities of 10 ⁇ 10 –10 ⁇ 12 M for a standard 50 kDa protein which encodes one to ten radiolabeled methionine residues (82).
  • the approach, described herein, to identify high affinity binding sequences or target sites for drug development can also be used in high throughput assays to screen for small molecules from combinatorial libraries.
  • one or more target sites are synthesized in each of the 96-well plates as described earlier.
  • the same high affinity binding sequence is synthesized in all of the wells.
  • a radiolabeled protein B from an in vitro transcription-translation mix for example, is added and allowed to incubate as indicated above.
  • bound protein B is eluted with SDS-sample buffer.
  • Wells containing radiolabeled protein B indicates that the drug had no effect on the binding between the high affinity binding sequence and protein B.
  • that drug has inhibited the interactions between the high affinity binding sequence on A and protein B.
  • the latter drug is a good LEAD compound.
  • the present invention also provides quantitative assays to measure the protein-protein interaction and the modulation thereof by compounds.
  • MDR multidrug resistant tumor cells
  • P-gp MDR1 P-glycoprotein
  • MRP1 multidrug resistance-associated protein
  • P-gp1 Although, the structure of P-gp1 remains a matter speculation (91), cumulative topological evidence suggest a tandemly duplicated structure of six transmembrane domains and a large cytoplasmic domain encoding an ATP binding sequence (58, 68). The two halves of P-gp1 are linked by a stretch of 90 residues rich in polar or charged amino acids, termed the “linker domain.”
  • the P-gp gene family is made up of three structurally similar isoforms in rodents (classes I, II, and III) and two isoforms in humans (classes I and III) (20). Gene transfer studies suggest functional differences among these structurally similar isoforms. For example, only the P-gp isoforms of classes I and II confer the MDR phenotype (25, 111), while the class III isoforms do not (11, 98). The class III isoforms mediate the transfer of phosphatidylcholine from the inner to the outer leaflet of the plasma membrane (i.e., “flipase”) (92, 100).
  • P-gp distribution is restricted mainly to tissues with secretory functions (79, 116). Its polarized localization to apical surfaces facing a lumen in the adrenal gland, liver, kidney intestine suggests a normal transport or detoxification mechanism. Moreover, hematopoietic stem cells and specific lymphocyte subclasses also express high levels of P-gp (49).
  • the normal function or substrate(s) of the classes I and II remain undefined; however, the disruption of the class I or/and II genes from the mouse genome results in the accumulation of cytostatic drugs or lipophilic compounds in most normal tissues, but more strikingly in the brain (99, 100). Based on these results it is speculated that the normal function of P-gp (the class I and II or the MDR causing P-gp) is detoxification similar to that seen in MDR cells, especially at the blood brain barrier (57).
  • MDR-reversing agents include verapamil, quinidine, ivermectin, cyclosporins, and dipyrimadol analogues to name but few (34, 38).
  • Clinical trials using MDR-reversing agents have shown some response in tumors that were otherwise non-responsive to chemotherapy (23, 44, 117).
  • MDR-reversing agents i.e., cyclosporin A and its non-immunosuppressive analog PSC833; however toxic effects have also been seen with cyclosporins (101, 115).
  • P-gp was shown to be a substrate for protein kinases C and A (2, 9). Moreover, it has been demonstrated that agents which modulate protein kinase C activity, modulate P-gp phosphorylation and its MDR-mediate phenotype (7, 13). In one study (31), PMA phorbol ester (a protein kinase C activator) was shown to increase the MDR phenotype and drug efflux in MCF7 breast cancer cells. In another study (6), sodium butyrate treatment of SW620 human colonic carcinoma cells was shown to result in a large increase in P-gp expression without a concomitant increase in drug-resistance or efflux.
  • the latter assay is based on the novel understanding of protein interactions provided by the present invention.
  • the results show hereinbelow that three sequences in the linker domain bind to proteins with apparent molecular masses of ⁇ 80 kDa, 57 kDa and 30 kDa.
  • Purification and partial N-terminal amino acid sequencing of the 57 kDa protein showed that it encodes the N-terminal amino acids of ⁇ and ⁇ -tubulins.
  • this domain is bound specifically to proteins; ii) the specifically binding proteins can be formally identified; and iii) the sequence responsible for the specific binding of these proteins formally identified (together with the interacting domain of this binding protein, if derived).
  • Pre-derivatized plastic rods, active ester and polypropylene trays were purchased from Cambridge Research Biochemicals (Valley Stream, N.Y.). Peptides were synthesized on solid polypropylene rods as previously described (36, 37). Briefly, the F-moc protecting group on the prederivatized polypropylene rods as solid support (arranged in a 96-well formate) was removed by incubation with 20% (v/v) piperidine in dimethylformamide (DMF) for 30 minutes with shaking. Following the deprotection of the ⁇ -alanine spacer on the polypropylene rods, Fmoc protected amino acids were dissolved in HOBt/DMF and added to the appropriate wells containing deprotected rods.
  • DMF dimethylformamide
  • Coupling of amino acids was allowed to take place for 18 hours at room temperature after which the rods were washed in DMF (1 ⁇ 2 minutes), methanol (4 ⁇ 2 minutes), and DMF (1 ⁇ 2 minutes).
  • the coupling of the second amino acid required the deprotection of the F-moc amino protecting group of the first amino acid and incubation of the rods with the second preactivated F-moc-protected amino acids (pentafluorophenyl derivatives).
  • the reaction was allowed to proceed for 18 hours, and the rods were removed and washed as indicated above. The same steps were repeated for each amino acid coupling until the sixth amino acid was coupled.
  • the F-moc N-terminal protecting group was removed with 20% piperidine/DMF and the free amino group acetylated for 90 minutes in an acetylation cocktail containing acetic anhydride:diisopropylethylamine (DIEA):DMF (50:1:50 v/v/v).
  • DIEA acetic anhydride:diisopropylethylamine
  • the side chain protecting groups of the N-terminal acetylated hexapeptides onto the polypropylene rods were removed by incubation in a cleavage mixture containing trifluoroacetic acid:phenol:ethandithiol (95:2.5:2.5 v/v/v) for 4 hours at room temperature.
  • the rods were washed with dichloromethane (DCM) and neutralized in 5% (v/v) DIEA/DCM.
  • DCM dichloromethane
  • the deprotected peptide-coupled rods were washed in DCM, methanol and vacuum dried for 18 hours.
  • CEM and CEM/VLB 1.0 Drug sensitive (CEM) and resistant (CEM/VLB 1.0 ) cells were cultured in ⁇ -MEM media supplemented with 10% fetal calf serum (Hyclone, Inc.) as previously described (8). All cells were examined for Mycoplasma contamination every three months using the Mycoplasma PCR kit from Stratagene Inc. (San Diego, Calif.). For metabolic labeling of cells, CEM or CEM/VLB 1.0 cells at 70–80% confluency were metabolically labeled with [ 35 S] methionine (100 ⁇ Ci/ml) for 6 hours at 37° C. in methionine-free ⁇ -MEM media.
  • hypotonic buffer 10 mM KCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl, pH 7.4
  • protease inhibitors 2 mM PMSF, 3 ⁇ g/ml Leupeptin, 4 ⁇ g/ml pepstatin A and 1 ⁇ g/ml aprotinin
  • Cells were lysed by homogenization in a hypotonic buffer and the cell lysate was sequentially centrifuged at 6000 ⁇ g for 10 minutes.
  • the supernatant was removed and made 0.5 M NaCl final concentration from a stock solution of 4 M NaCl.
  • the cell lysate was incubated on ice for 30 minutes. The sample was mixed and brought back to 0.1 M NaCl final concentration. The cell lysate was centrifuged for 10 minutes at 15,000 ⁇ g at 4° C. The latter supernatant was removed and recentrifuged at 100,000 ⁇ g for 60 minutes in a Beckman ultracentrifuge using SW55 rotor. The amount of protein in the above samples was determined by the method of Lowry (69).
  • [ 35 S] methionine labeled proteins from total cell lysate were mixed with equal volume of 3–6% BSA in phosphate buffered saline (PBS) and incubated with overlapping hexapeptides covalently fixed to polypropylene rods.
  • the peptides and total cell lysate were incubated overnight at 4° C.
  • the rods were then removed and washed four times in PBS.
  • the bound proteins were eluted by incubating the peptide-fixed rods in 1 ⁇ SDS sample buffer for 60 minutes at room temperature with shaking.
  • the peptides-fixed rods were regenerated by incubation in PBS, containing 2% SDS and 1 mM ⁇ -mercaptoethanol at 65° C. in a sonicator for 30 minutes. Following the latter incubation, the rods were washed for five minutes in 65° C. ionized water and two minutes in 65° C. methanol. The peptides-fixed rods are now ready for the next round of screening.
  • [ 35 S] methionine labeled proteins from total cell lysate were mixed with equal volume of 3% BSA in phosphate buffered saline containing KCl (300 mM to 1200 mM), SDS (0.12% to 2%), or CHAPS (20 mM to 160 mM) and incubated with covalently fixed peptides as described above.
  • Protein fractions (100–150 ⁇ l) were resolved on SDS-PAGE using the Laemmli gel system (62). Briefly, proteins were dissolved in 1 ⁇ solubilization sample buffer I (62.5 mM Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 10% (w/v) glycerol and 5% ⁇ -mercaptoethanol) and samples were electrophoresed at constant current. Gel slabs containing the resolved proteins were fixed in 50% methanol and 10% acetic acid. Polyacrylamide gels containing [ 35 S] methionine proteins were exposed to Kodak x-ray film following a thirty-minute incubation in an AmplifyTM solution (Amersham Inc.).
  • proteins were transferred to nitrocellulose membrane in Tris-glycine buffer in the presence of 20% methanol for Western blot analysis according to the procedure of Towbin et al. (108).
  • Nitrocellulose membrane was incubated in 5% skim milk/PBS prior to the addition of anti- ⁇ or anti- ⁇ tubulin monoclonal antibodies (0.5 ⁇ g/ml in 3% BSA; Amersham, Inc.). Following several washes with PBS, the nitrocellulose membrane was incubated with goat anti-mouse peroxidase-conjugated antibody and immunoreactive proteins were visualized by chemiluminescence using ECL method (Amersham, Inc.).
  • the 57 kDa associated protein was purified using a block of polypropylene rods with two high affinity binding peptides. Briefly, the peptide-fixed rods were incubated with total cell lysate as indicated above; however, in this case the carrier substance was gelatin (1%). The bound proteins were eluted in 100 mM phosphate buffer, pH 7.4 containing 2% SDS and 0.1% ⁇ -mercaptoethanol. The eluted proteins were precipitated by mixing with 9 volumes of ice cold ethanol and incubated at ⁇ 20° C.
  • P-gp is a tandemly duplicated molecule made up of two halves with each encoding for six transmembrane domains and an ATP binding domain. The two halves of P-gp are linked by a linker domain. Of the 90 amino acids that make up the linker domain, 32 amino acid are either positively or negatively charged at physiological pH. While P-gp phosphorylation sites appear to have relevance to P-gp function, the function of the linker domain of P-gp remains unknown. To identify and dissect the role of this domain in MDR, the overlapping peptides method of the present invention was used. A novel approach was developed to isolate interacting proteins using overlapping synthetic hexapeptides.
  • FIG. 5 shows the amino acid sequences of the linker domain of HP-gp 1 and HP-gp 3.
  • the two linker domains of HP-gp1 and HP-gp3 share 41% amino acid sequence identity and 66% sequence homology.
  • Overlapping hexapeptides were synthesized in parallel on derivatized polypropylene rods as previously described (36, 37). 92 and 90 hexapeptides were synthesized to cover the entire linker sequence of HP-gp1 and HP-gp3, respectively. The hexapeptides remain covalently attached to the polypropylene rods.
  • FIG. 6 shows the proteins specifically bound to the 92 overlapping hexa-peptides from HP-gp1 linker sequence.
  • hexapeptides 3–10 bound to ⁇ 210 kDa protein that was not detected previously in the absence of CHAPS.
  • hexapeptides 16–20 which did not bind any proteins in the absence of CHAPS, bound to the same high molecular weight protein ( FIG. 7 ).
  • Peptides 40–60 bound more strongly to several low molecule weight proteins ( ⁇ 45–25 kDa) in the presence of CHAPS.
  • the hexapeptides 80–89 bound to two other proteins in addition to the 57 kDa protein.
  • the solubilization of membrane proteins in 10 mM CHAPS show binding to other proteins not seen in the absence or 10 mM CHAPS.
  • 10 mM CHAPS allows integral membrane proteins to interact with the various hexapeptides of HP-gp 1 linker domain.
  • CHAPS exposes new domains that in turn allows for binding to hexapeptides of HP-gp1 linker domain.
  • some of the lower molecular weight proteins that bound to hexapeptides 40–60 and 80–89 may be degradation products of the 57 kDa protein ( FIG. 8 ).
  • HP-gp 1 and HP-gp 3 are encoded by two isoforms, HP-gp 1 and HP-gp 3 (or mdr 1 and mdr 3; (20)).
  • HP-gp 1 confers an MDR phenotype.
  • HP-gp 1 and 3 share about 80% amino acid sequence homology (111); the linker domain is the most variable domain among the two isoforms with 66% amino acid sequence homology.
  • overlapping hexapeptides encoding HP-gp 3 linker domain were synthesized on polypropylene rods and their binding to soluble proteins was examined as indicated above.
  • the first and third regions of HP-gp 3 linker domain share considerable sequence identity with the first and third regions of HP-gp 1 linker domain ( FIG. 10 ). Hence, it is not surprising that the same hexapeptides bound to the same protein.
  • the second region of HP-gp 1 and HP-gp 3 linker domains are different ( FIG. 10 ). Consequently, although both the HP-gp1 and HP-gp3 sequences bound to a 57 kDa, the region of interaction between HP-gp 3 and the 57 kDa protein is larger than that of HP-gp 1 ( FIG. 6 and FIG. 9 ).
  • a comparison of the amino acid sequences from HP-gp 1 and HP-gp 3 binding hexapeptides is shown in FIG. 10 .
  • FIG. 11 shows the binding of these two peptides to total cell lysate from [ 35 S] methionine metabolically labeled cells.
  • Both hexapeptides bound specifically to the 57 kDa protein and another protein of an apparent molecular mass of ⁇ 41 kDa.
  • longer incubation times of the total cell lysate led to an increase in the level of the 41 kDa protein ( FIG. 11 ).
  • the 41 kDa band is likely a degradation product of the 57 kDa protein.
  • FIG. 12 shows the effects of no blocking carrier, 1% gelatin and 0.3% or 3% BSA on the binding of the hexapeptides to the 57 kDa protein.
  • the results of this experiment were surprising in that no carrier protein was required to reduce the unspecific binding ( FIG. 12 ).
  • the latter established binding conditions were used to isolate large amounts of 57 kDa protein that bound to several copies of hexapeptides 658 RSSLIR 663 (SEQ ID NO: 7) and 669 SVRGSQ 674 (SEQ ID NO: 8).
  • FIG. 13 shows purified 57 kDa protein on SDS-PAGE stained with Coomassie blue.
  • the latter purified protein was transferred to PVDF membrane and stained with Ponceau S to localize the position of the 57 kDa protein.
  • the Ponceau S-stained band that migrated with the expected molecular mass was cut out and used for direct N-terminal sequencing (33).
  • the first seven rounds of Edman degradation showed two sequences of MREVISI (SEQ ID NO: 10) and MREIVHI (SEQ ID NO: 11). These two sequences differed only by three amino acids (VIS instead of IVH).
  • the overlapping peptide-based method of the present invention provides the proof of principle to the hypothesis which states that the region between two interacting proteins consists of high affinity binding sequences and repulsive sequences as well as the fact that such a method can be used efficiently and successfully to identify and characterize domains and sequences of interacting proteins.
  • the balance of high affinity and repulsive forces determine whether two proteins will form stable complex.
  • the use of short overlapping peptides allows the identification of such high affinity binding sequences between bait and prey proteins.
  • the rationale for using short overlapping peptides to isolate high affinity binding sequences is essential to the success and efficiency of the proof of the principle described herein.
  • larger peptides could contain both high affinity and repulsive binding sequences in one peptide sequence such that the net force of interaction is negative.
  • the use of overlapping peptides that differ by one amino acid from the previous or next peptide reduces the possibility of unspecific binding.
  • overlapping peptides often demonstrate a peak in the binding affinity of various peptides (see FIGS. 7 and 4 ).
  • the skilled artisan will understand that longer overlapping peptides could also be used.
  • such larger peptides increase the risk of missing the identification of interacting proteins due to a change in the balance between high affinity and repulsive amino acids.
  • the binding of 57 kDa protein to three different regions in HP-gp1 and HP-gp3 linker domains is consistent with the herein proposed hypothesis to explain protein interactions (see principle of protein-protein interactions).
  • the high affinity binding domains vary in sizes from 10–26 amino acids in length. In the case of HP-gp1 and HP-gp3 linker domains, two of the three high affinity binding domains shared considerable sequence identity.
  • the third high affinity binding region of the linker domains 658 SRSSLIRKRSTRRSVRGSQA 677 (SEQ ID NO: 2) versus 648 KAATRMAPNGWKSRLFRHSTQKNLKNS 674 (SEQ ID NO: 5)) shared no homology in their primary amino acid sequence.
  • 57 kDa protein binds to the polypropylene rods or their derivatized moieties is unlikely since all other rods which are similarly derivatized did not bind the 57 kDa protein.
  • other proteins with apparent molecular masses of ⁇ 80 kDa and 30 kDa also bound to some of the hexapeptides in the linker domains.
  • HP-gp1 or HP-gp3 binding to tubulin has been shown to interact with several membrane proteins (42, 50, 81, 86).
  • HP-gp1 or HP-gp3 interactions with tubulin and possibly microtubules maybe an example of the membrane-skeleton fence model (56).
  • a small fraction of membrane receptors seem to be fixed to the underlying cytoskeleton (95).
  • increase in the stability and expression of P-gp in rat liver tumors in vivo are associated with similar increases in the stability of several cytoskeleton proteins, including ⁇ -tubulin, ⁇ -actin, and cytokeratins 8/18 (64). Work is in progress to determine the functional significance of P-gp interactions with tubulin in vivo.
  • Annexin I a soluble and membrane associated protein, as opposed to P-glycoprotein, a strictly transmembrane protein.
  • Annexin is thus structurally and functionally different from P-glycoprotein.
  • Annexin I is a member of a large family of intracellular soluble and membrane associated proteins that bind phospholipids in a reversible and calcium-dependent manner.
  • Various members of the Annexin family have been implicated in a number of different intracellular processes including vesicular trafficking, membrane fusion exocytosis, signal transduction, and ion channel formation and drug resistance.
  • the method of the present invention was set out to identify its interacting proteins and the precise amino acid sequences that mediate Annexin I interactions thereto.
  • overlapping peptides corresponding to the entire amino acid sequence of Annexin I were synthesized on a solid support as described above.
  • overlapping heptapeptides, as opposed to hexapeptides were used.
  • the peptides were then incubated with total cellular proteins isolated from MCF7 breast tumor cells that were metabolically labeled with [ 35 S] methionine. Following several washes, the bound proteins were eluted and resolved on SDS-PAGE as outlined above.
  • the present invention is shown to enable the simple and efficient identification of high affinity protein interaction as well as enabling the simultaneous identification of the precise amino acid sequence of at least one of the interacting partners.

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